Quantification of Ca21 uptake in the sarcoplasmic reticulum of trout ventricular myocytes

Hove-Madsen, Leif, Anna Llach, and Lluis Tort. Quantification of Ca21 uptake in the sarcoplasmic reticulum of trout ventricular myocytes. Am. J. Physiol. 275 (Regulatory Integrative Comp. Physiol. 44): R2070–R2080, 1998.—We measured Ca21 uptake by the sarcoplasmic reticulum (SR) in trout ventricular myocytes, measuring indo 1 fluorescence in permeabilized cells or ionic currents in single myocytes subjected to voltage clamp. Titration of the SR Ca21 pumps with thapsigargin gave a pump site density of 454 pmol/mg cell protein. Lowering the temperature from 20°C to 10 or 5°C reduced the SR Ca21 uptake rate in permeabilized myocytes by 50 and 63%, respectively. Surprisingly, Ca21 leak from the SR also decreased with decreasing temperatures. Exposure of single myocytes to 10 mM caffeine (Caf) induced a cell contracture and an inward ionic current. Neither contracture nor current decreased significantly after rest periods of 120 and 320 s. The inward current was due to Ca21 extrusion by the Na1/Ca21 exchanger (NCX), and the time integral of the exchange current (INCX) was used to calculate the SR Ca21 content. This gave a steady-state SR Ca21 content of 22.5 6 2.8 amol Ca21/pF or 750 μM. When the SR was loaded by depolarizing the cell to 150 mV, the Ca21 content increased with increasing length of the depolarization, reaching a maximum of 52.0 6 5.9 amol Ca21/pF. When the cell was depolarized to different voltages for 3 s, a subsequent Cafinduced INCX increased with increasing voltage. At 1100 mV, the Ca21 content was 36.6 6 3.8 amol/pF, giving a maximal SR Ca21 uptake rate of 12.2 6 1.2 amol Ca21 ·pF21 ·s21 or 417 μM/s. We conclude that maximal SR Ca21 content and Ca21 uptake rates can be estimated using specific SR Ca21 loading protocols. Contrary to the general assumption that contraction in lower vertebrates depends largely on transsarcolemmal Ca21 fluxes, we found that although the L-type Ca21 current is insufficient to fully activate contraction, the SR is capable of participating in the regulation of the cytosolic Ca21 during the excitation-contraction coupling in trout ventricular myocytes.